JP2832760B2 - Sterilization method of immobilized enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for sterilizing an immobilized enzyme by using both an ultrasonic treatment and an ultraviolet irradiation treatment. By using the sterilization method of the present invention, microorganisms adhering to the immobilized enzyme can be sterilized without reducing the enzyme activity.

[0002]

2. Description of the Related Art Conventionally, immobilized enzymes have been widely used for producing or improving substances. However, if the immobilized enzyme is used continuously for a long period of time, microorganisms such as bacteria attached to the immobilized enzyme will proliferate, and as a result, problems such as microbial contamination of the product and reduced activity of the immobilized enzyme will occur. come.

In order to solve such problems, it has been proposed to use a bactericide or a detergent when washing the immobilized enzyme. For example, a method in which an immobilized enzyme is sterilized by contact with a dilute aqueous solution of a substituted diethylenetriamine such as dioctyldiethylenetriamine (Japanese Patent Laid-Open No. 56-9 / 1981)
2791), a method of immersing the immobilized enzyme contaminated with microorganisms in polyhydric alcohol for sterilization (JP-A-59-17587).
9), a method in which the immobilized enzyme is treated with a mixed solution containing laurylbenzenesulfonate and ethanol and washed and sterilized (JP-A-59-98689). However, the method of washing and disinfecting these immobilized enzymes with disinfectants and detergents requires a long time for washing and disinfection, or the washing and disinfecting effect is not always sufficient, and the activity of the immobilized enzymes is reduced. There is a problem that various solutions have to be taken, such as an operation of completely removing the detergent and the bactericide remaining on the immobilized enzyme after washing and sterilization.

[0004] Further, the intensity of irradiation of the immobilized enzyme with ultraviolet light is set to 0.1.
A method for sterilizing an immobilized enzyme, which irradiates at 5 mW / cm ² or less to kill microorganisms adhering to a carrier without lowering the activity of the immobilized enzyme (Japanese Unexamined Patent Publication No. 1-28455)
4) is disclosed. In order to obtain a desired sterilizing effect in this method, it is necessary to irradiate the entire surface of the immobilized enzyme with ultraviolet rays stably and uniformly, and a specific sterilizing apparatus is required. The applicant of the present invention pays attention to such a point, and repeats the operation of rotating and / or raising / lowering the immobilized enzyme in the washing / sterilization reaction tank, and irradiates the immobilized enzyme with ultraviolet light from thereover (Japanese Patent Application No. Hei. -43893).

[0005]

DISCLOSURE OF THE INVENTION The present inventors have intensively studied a method for increasing the bactericidal effect by ultraviolet irradiation without reducing the activity of the immobilized enzyme. Then, when the immobilized enzyme used for a long period of time was irradiated with ultraviolet rays and its bactericidal effect was measured, it was found that the bactericidal power which should originally be 10 ⁴ to 10 ⁵ is actually only about 10 ¹ . Therefore, the cause is examined, (a) that the microorganisms adhering to the immobilized enzyme overlap, (b) that the immobilized enzyme is covered with a substrate or a hydrolyzate thereof in a layer or film, It was assumed that ultraviolet rays did not reach the microorganisms due to such factors.

Next, as a result of studying the use of ultrasonic treatment in combination to remove these obstacles, (1) the ultraviolet irradiation treatment alone has a sterilizing power of about 10 ¹ , and even if the ultraviolet irradiation amount is increased, the sterilizing power is increased. (2) The ultrasonic treatment alone has a bactericidal power of about 10 ³ after the treatment for 30 minutes, the bactericidal power does not increase even if the treatment time is extended, and only the cleaning effect is obtained. ) After performing the ultrasonic treatment, the bactericidal power is sharply increased by performing the ultraviolet irradiation treatment, and the bactericidal power is about 10 ⁶ when the ultraviolet irradiation amount is 4000 mWs / cm ^2. (4) When the ultrasonic treatment and the ultraviolet irradiation treatment are used in combination, the longer the ultrasonic treatment, the longer the sterilization effect is obtained.

[0007] From these findings, there is a limit to the bactericidal activity of the ultraviolet irradiation treatment alone because (a) microorganisms overlap,
(B) due to being coated with a substrate or the like,
When the ultrasonic treatment and the ultraviolet irradiation treatment are used in combination, it has been found that the obstructive factors are eliminated, the ultraviolet light reaches the immobilized enzyme, and the bactericidal effect can be enhanced, and the present invention has been completed. It has also been found that, when performing the ultrasonic treatment, the disinfection effect can be further enhanced by performing the treatment in a liquid containing a surfactant. Therefore,
An object of the present invention is to provide a method for disinfecting microorganisms attached to an immobilized enzyme without reducing the enzyme activity.

[0008]

A feature of the present invention resides in that, when sterilizing an immobilized enzyme, an ultrasonic treatment and an ultraviolet irradiation treatment are used in combination. Another object of the present invention is to perform the treatment in a liquid containing a surfactant when performing the ultrasonic treatment. By performing such treatment, microorganisms adhering to the immobilized enzyme can be sterilized without reducing the enzyme activity.

[0009] The sterilization method of the present invention can be applied to any immobilized enzyme. As these immobilized enzymes, enzymes such as lactase, glucose isomerase, and protease are chemically bonded to a water-insoluble carrier such as chitosan, chitin, resin, or encapsulated in cellulose triacetate, polyacrylamide gel, or the like. Can be exemplified. These immobilized enzymes can be treated with ultrasonic waves and irradiated with ultraviolet rays regardless of whether they are packed in a column or the like, diffused into a stirred tank, or removed from those devices. The treatment can be used in combination.

[0010] The ultrasonic treatment is performed by using an ultrasonic vibrator placed in an apparatus in which the immobilized enzyme is filled or diffused, or by removing the immobilized enzyme from the apparatus and using an ultrasonic cleaner for several minutes to 6 hours.
Perform for about 0 minutes. At this time, if a surfactant is added to the liquid to be subjected to the ultrasonic treatment, the cleaning effect can be further increased. Before the ultrasonic treatment, the liquid may be treated with a liquid containing a surfactant. The ultrasonic treatment in the solution containing a surfactant in the present invention may be any of these cases. The surfactant used here is not particularly limited, and glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, lecithin and the like are used. The glycerin fatty acid ester may be a monoglycerin fatty acid ester or a polyglycerin fatty acid ester. For example, glycerin fatty acid ester, glycerin acetic acid fatty acid ester, glycerin lactic acid fatty acid ester, glycerin citrate fatty acid ester, glycerin succinic acid fatty acid ester, glycerin diacetyl tartaric acid ester, glycerin acetate, and the like. Span 80), sorbitan monolaurate (Span 20) and the like. As sucrose fatty acid esters, esters containing palmitic acid and stearic acid as fatty acids are used. These surfactants are usually used by dissolving in 0.5 to 2.0% by weight of distilled water. Among these surfactants, polyglycerin fatty acid ester is an emulsifier widely used as a food additive, and is used in the present invention because it has high safety, enhances the washing effect and does not decrease the activity of the immobilized enzyme. It is particularly suitable for

The ultraviolet irradiation treatment is carried out at an illuminance and time sufficient to kill microorganisms adhering to the immobilized enzyme. Usually, the irradiation is carried out at an illuminance of about 10 mW / cm ² for 100 seconds to 2,000 seconds, and the irradiation amount is 1,000 to 20,000 mWs / cm ^2. cm ²
It is preferable to perform so that Especially the irradiation dose is 300
The effect is obtained at 0 mWs / cm ² or more.

As for the combination of the ultrasonic treatment and the ultraviolet irradiation treatment, the ultraviolet treatment may be performed after the ultrasonic treatment, or the ultrasonic treatment and the ultraviolet irradiation treatment may be performed simultaneously in parallel. At this time, a conventionally known ultraviolet irradiation method or processing apparatus, for example, JP-A 1-2284
54 and Japanese Patent Application No. 2-43893 can be used in combination with ultrasonic treatment.

Further, by washing the immobilized enzyme with distilled water or a buffer before and after the ultrasonic treatment or the ultraviolet irradiation treatment, the substrate adhering to the immobilized enzyme, its decomposed product,
Alternatively, it is desirable to remove microorganisms.

[0014] The sterilization method of the present invention specifically includes sterilization of an immobilized enzyme obtained by immobilizing lactase used in decomposing lactose in milk into glucose and gactose on a carrier, or lactose, whey, skim milk and the like. Can be used for sterilization of an immobilized enzyme obtained by immobilizing β-galactosidase used for producing oligosaccharides from a carrier on a carrier. Furthermore, it can be used for sterilization of an immobilized enzyme in which a protease used for decomposing a protein into peptides or free amino acids is immobilized on a carrier.

[0015]

The effects of the present invention will be described with reference to test examples.

Test Example 1 Protease S (manufactured by Amano Pharmaceutical Co., Ltd.) was used as an enzyme, cross-linked with glutaraldehyde, immobilized on chitosan beads (trade name: Chitopearl, manufactured by Fuji Boseki Co., Ltd.), and immobilized. An enzyme (immobilized protease S) was prepared. This immobilized protease S was repeatedly used for the casein hydrolysis treatment. As a result, the contaminated microorganisms were grown to 2.7 × 10 ⁶ CFU / ml and subjected to a washing and sterilization test. did.

The immobilized protease S contaminated as described above is treated with an ordinary ultrasonic cleaner having an output of about 100 W for 3 hours.
Ultrasonic treatment was performed for 0 to 60 minutes, and at the same time, ultraviolet irradiation was performed from above. Table 1 shows the results. The number of remaining microorganisms was significantly reduced by using both the ultrasonic treatment and the ultraviolet irradiation treatment as compared with the control. In particular, when the ultraviolet irradiation amount was 3000 mWs / cm ² or more, almost no microorganisms remained. From these results, it was found that when ultrasonic treatment and ultraviolet irradiation treatment were used in combination for sterilization of the immobilized enzyme, the attached microorganisms could be sterilized without reducing the enzyme activity. This bactericidal effect was remarkable when the irradiation amount of ultraviolet rays was 3000 mWs / cm ² or more.

[0017]

[Table 1]

[0018]

Test Example 2 Using immobilized protease S contaminated by the same treatment as in test example 1, the following samples were prepared in order to confirm the effect of the surfactant.
Was measured for residual activity.

(1) Sample 1 Spent immobilized protease S was output in distilled water at a power of 100 W
Ultrasonic treatment with a commonly used ultrasonic cleaner. (2) Sample 2 Normally, about 100 W of output was used in distilled water to which 1% by weight of glycerin fatty acid ester / SY Glister ML750 (trade name, manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) was added using used immobilized protease S as a surfactant. Ultrasonic treatment with an ultrasonic cleaner. (3) Sample 3 Normally, about 100 W of output was used in distilled water to which 1% by weight of glycerin fatty acid ester / SY Glister ML750 (trade name, manufactured by Sakamoto Yakuhin Kogyo) was added using used immobilized protease S as a surfactant. Ultrasonic treatment with an ultrasonic cleaner, and simultaneous irradiation with ultraviolet light from above.

The UV illuminance was 5 mW / cm ² on average. The results are shown in FIG. As shown in this figure, it can be seen that the surfactant hardly reduces the enzyme activity. The reason that the residual activity of the immobilized protease S became higher than the initial activity in the ultrasonic treatment for 60 minutes was that the hydrolyzate and microorganisms attached to the surface of the immobilized protease S were removed, and the contact with the substrate became smooth. It is because.

[0021]

Test Example 3 Using immobilized protease S contaminated by the same treatment as in test example 1, in order to confirm the effect of the surfactant, the following sample was prepared and immobilized protease S
A cleaning sterilization test was performed by counting the number of microorganisms remaining on the surface.

(1) Sample 1 Spent immobilized protease S was output in distilled water at a power of 100 W
Ultrasonic treatment with a commonly used ultrasonic cleaner and irradiation of ultraviolet light from above in parallel with the ultrasonic treatment. The UV illuminance was adjusted to be about 5 mW / cm ² . (2) Sample 2 About 100 W in distilled water to which 0.5% by weight of glycerin fatty acid ester / SY Glister ML750 (trade name, manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) was added using used immobilized protease S as a surfactant. Ultrasonic treatment with a commonly used ultrasonic cleaner, and simultaneous irradiation with ultraviolet light from above. The UV illuminance was adjusted to be about 5 mW / cm ² . (3) Sample 3 Normally, about 100 W of output was used in distilled water to which 1% by weight of glycerin fatty acid ester / SY Glister ML750 (trade name, manufactured by Sakamoto Yakuhin Kogyo) was added using used immobilized protease S as a surfactant. Ultrasonic treatment with an ultrasonic cleaner, and simultaneous irradiation with ultraviolet light from above. The UV illuminance was adjusted to be about 5 mW / cm ² . In each case, the processing time was 0 to 60 minutes.

FIG. 2 shows the results. As can be seen from the table, the surfactant exhibits a bactericidal effect according to the amount of the bactericidal treatment with the passage of the germicidal treatment time.

Next, the present invention will be specifically described with reference to examples of the present invention.

Example 1 A column having a diameter of 6 cm and a length of 18 cm was filled with 30
After filling with 0 ml of the immobilized protease S, a hydrolyzate was prepared by continuously passing 10% casein in a down flow at a reaction temperature of 40 ° C. and SV = 3. With the passage of time, the growth of the bacterium began, and milk protein adhered to the immobilized protease S and bacterial contamination occurred. The residual enzyme specific activity after passing the solution for 12 hours was 70%, and the number of microorganisms was of the order of 10 ⁶ . At this time, the immobilized protease S
After removing the solid content by washing with a phosphate buffer of pH 7.0, the immobilized protease S was transferred to a sterilization tank, and irradiated with 5 mW / cm ² ultraviolet rays for 60 minutes while rotating the sterilization tank. Simultaneously, an ultrasonic wave was emitted from an ultrasonic oscillator having an output of 100 W. The immobilized protease S thus treated was washed with a sterilized phosphate buffer of pH 7.0, the residual enzyme specific activity was restored to 100%, and the number of adherent microorganisms was 0.0 × 10 ¹ . there were. This immobilized protease S was packed again in the column and used for the hydrolysis of casein.

[0025]

Example 2 30 columns in a column of 6 cm in diameter and 18 cm in length
After filling with 0 ml of the immobilized protease S, a hydrolyzate was prepared by continuously passing 10% casein in a down flow at a reaction temperature of 40 ° C. and SV = 3. With the passage of time, the growth of the bacterium began, and milk protein adhered to the immobilized protease S and bacterial contamination occurred. The residual enzyme specific activity after passing the solution for 12 hours was 70%, and the number of microorganisms was of the order of 10 ⁶ . At this time, the immobilized protease S
Was taken out and washed with a phosphate buffer of pH 7.0 to remove solids, and then 1% by weight of glycerin fatty acid ester / SY Glister ML750 (trade name, manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) as a surfactant. Ultrasonic waves were radiated using a 100 W output ultrasonic oscillator in distilled water to which% was added, and 5 mW / cm ² ultraviolet rays were irradiated for 60 minutes while rotating the sterilizing tank. The immobilized protease S thus treated is washed with a sterile-treated phosphate buffer of pH 7.0, the remaining enzyme specific activity is 100%, and the number of adherent microorganisms is 0.0 × 10 ¹ or less. there were. This immobilized protease S was packed again in the column and used for the hydrolysis of casein.

[Brief description of the drawings]

FIG. 1 shows the effect of the cleaning and sterilizing treatment of Test Example 2 on enzyme activity.

[Explanation of symbols]

─ ○ ─ Ultrasonic treatment 併 用 Combination of ultrasonic treatment and surfactant ─ □ ─ Combination of ultrasonic treatment, surfactant and UV irradiation treatment

FIG. 2 shows the cleaning / sterilization assisting effect of the surfactant of Test Example 3.

[Explanation of symbols]

 ─ □ ─ No surfactant added ─ △ ─ 0.5% surfactant added ─ ○ ─ 1% surfactant added

Continuation of the front page (56) References JP-A-1-228454 (JP, A) JP-A-63-237787 (JP, A) JP-A-61-12280 (JP, A) JP-A-3-247269 (JP) , A) Actually open 63-145500 (JP, U) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 11/00-11/18

Claims (4)

(57) [Claims] 1. A method for disinfecting an immobilized enzyme, comprising using an ultrasonic treatment and an ultraviolet irradiation treatment together with the immobilized enzyme to sterilize microorganisms adhering to the immobilized enzyme. 2. The method for sterilizing an immobilized enzyme according to claim 1, wherein the ultrasonic treatment is performed in a liquid containing a surfactant. 3. The method according to claim 2, wherein the surfactant is a glycerin fatty acid ester. 4. An ultraviolet irradiation treatment is performed at 3000 mWs / cm.
^The method for sterilizing an immobilized enzyme according to any one of claims 1 to 3, wherein the method is performed with ^two or more ultraviolet irradiation doses.